Inhibitors Of IKK-Beta Serine-Threonine Protein Kinase

ABSTRACT

Compounds of formula (IA) or (IB) are inhibitors of IkB kinase (IKK) activity, and are useful in the treatment of autoimmune and inflammatory diseases: 
     
       
         
         
             
             
         
       
     
     wherein R 7  is hydrogen or optionally substituted (C 1 -C 6 )alkyl; ring A is an optionally substituted aryl or heteroaryl ring of 5-13 ring atoms; Z is a radical of formula R-L 1 -Y 1 —(CH 2 ) z —, wherein: z is 0 or 1; R is a radical of formula (X) or (Y) 
     
       
         
         
             
             
         
       
     
     R 1  is a carboxylic acid group (—COOH), or an ester group which is hydrolysable by one or more intracellular esterase enzymes to a carboxylic acid group; R 6  is hydrogen, or optionally substituted C 1 -C 6  alkyl, C 3 -C 7  cycloalkyl, aryl or heteroaryl or —(C═O)R 3 , —(C═O)OR 3 , or —(C═O)NR 3  wherein R 3  is hydrogen or optionally substituted (C 1 -C 6 )alkyl; Y 1  is a bond, —(C═O)—, —S(O 2 )—, —C(═O)O—, —OC(═O)—, —(C═O)NR 3 —, —NR 3 (C═O)—, —S(O 2 )NR 3 —, —NR 3 S(O 2 )—, or —NR 3 (C═O)NR 4 —, wherein R 3  and R 4  are independently hydrogen or optionally substituted (C 1 -C 6 )alkyl, L 1  is a divalent linker radical of formula -(Alk 1 ) m (Q) n (Alk 2 ) p - wherein m, n, p, Q, Alk 1  and Alk 2  are as defined in the claims.

This invention relates to thiophene carboxamides characterised by thepresence in the molecule of an amino acid ester group, to compositionscontaining them, to processes for their preparation and to their use inmedicine as IKK inhibitors for the treatment of autoimmune andinflammatory diseases, including chronic obstructive pulmonary disease,asthma, rheumatoid arthritis, psoriasis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, multiple sclerosis, diabetes,atopic dermatitis, graft versus host disease, systemic lupuserythematosus. The compounds are also of use in the treatment ofproliferative disease states, such as cancers.

BACKGROUND OF THE INVENTION

The expression of many pro-inflammatory genes is regulated by thetranscriptional activator nuclear factor-kB (NF-kB). These transcriptionfactors have been suspected since their discovery to play a pivotal rolein chronic and acute inflammatory diseases. It now seems that aberrantregulation of NF-kB could also underlie autoimmune diseases anddifferent types of cancer.

Examples of genes dependent on the activation of NF-kB include: thecytokines tumor necrosis factor TNF-α, interleukin (IL)-6, IL-8 andIL-1β; the adhesion molecules E-selectin, intercellular adhesionmolecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1; and theenzymes nitric oxide synthase (NOS) and cyclooxygenase (COX)-2. NF-kBnormally resides in the cytoplasm of unstimulated cells as an inactivecomplex with a member of the IkB inhibitory protein family. However,upon cellular activation, IkB is phosphorylated by the IkB kinase (IKK)and is subsequently degraded. Free NF-kB then translocates to thenucleus where it mediates pro-inflammatory gene expression.

There are three classical IkB's: IkBα, IkBβ and IkBε; all of whichrequire the phosphorylation of two key serine residues before they canbe degraded. Two major enzymes IKK-α and IKK-β appear to be responsiblefor IkB phosphorylation. Dominant-negative (DN) versions of either ofthese enzymes (where ATP binding is disabled by the mutation of a keykinase domain residue) were found to suppress the activation of NF-kB byTNF-α, IL-1β and LPS. Importantly IKK-β DN was found to be a far morepotent inhibitor than IKK-α DN (Zandi, E Cell, 1997, 91, 243).Furthermore, the generation of IKK-α and IKK-β deficient miceestablished the requirement of IKK-β for activation of NF-kB byproinflammatory stimuli and reinforced the dominant role of IKK-βsuggested by biochemical data. Indeed it was demonstrated that IKK-α wasdispensable for NF-kB activation by these stimuli (Tanaka, M.; Immunity1999, 10, 421). Thus, inhibition of IKK-β represents a potentiallyattractive target for modulation of immune function and hence thedevelopment of drugs for the treatment of auto-immune diseases.

BRIEF DESCRIPTION OF THE INVENTION

This invention makes available a class of thiophene carboxamides whichare potent and selective inhibitors of IKK isoforms, particularly IKKβ.The compounds are thus of use in medicine, for example in the treatmentof a variety of proliferative disease states, such as conditions relatedto the hyperactivity of IKK, as well as diseases modulated by the NF-kBcascade. In addition, the compounds of the invention are useful for thetreatment of stroke, osteoporosis, rheumatoid arthritis and otherinflammatory disorders. The compounds are characterised by the presencein the molecule of an amino acid motif or an amino acid ester motifwhich is hydrolysable by an intracellular carboxylesterase. Compounds ofthe invention having the lipophilic amino acid ester motif cross thecell membrane, and are hydrolysed to the acid by the intracellularcarboxylesterases. The polar hydrolysis product accumulates in the cellsince it does not readily cross the cell membrane. Hence the IKKinhibitory activity of the compound is prolonged and enhanced within thecell. The compounds of the invention are related to the IKK inhibitorsencompassed by the disclosure in International Patent Application No. WO2004063186 but differ therefrom in that the present compounds have theamino acid ester motif referred to above.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided compound of formula(IA) or (IB), or a salt, N-oxide, hydrate or solvate thereof:

wherein

R₇ is hydrogen or optionally substituted (C₁-C₆)alkyl;

ring A is an optionally substituted aryl or heteroaryl ring or ringsystem of 5-13 ring atoms;

Z is a radical of formula R-L¹-Y¹—(CH₂)_(z)—, wherein:

-   -   R is a radical of formula (X) or (Y)

-   -   R₁ is a carboxylic acid group (—COOH), or an ester group which        is hydrolysable by one or more intracellular esterase enzymes to        a carboxylic acid group;    -   R₅ is hydrogen; or optionally substituted C₁-C₆ alkyl, C₃-C₇        cycloalkyl, aryl or heteroaryl or —(C═O)R₃, —(C═O)OR₃, or        —(C═O)NR₃ wherein R₃ is hydrogen or optionally substituted        (C₁-C₆)alkyl,    -   Y¹ is a bond, —(C═O)—, —S(O₂)—, —C(═O)O—, —OC(═O)—, —(C═O)NR₃—,        —NR₃(C═O)—, —S(O₂)NR₃—, —NR₃S(O₂)—, or —NR₃(C═O)NR₄—, wherein R₃        and R₄ are independently hydrogen or optionally substituted        (C₁-C₆)alkyl,    -   L¹ is a divalent radical of formula        -(Alk¹)_(m)(Q)_(n)(Alk²)_(p)- wherein        -   m, n and p are independently 0 or 1,        -   Q is (i) an optionally substituted divalent mono- or            bicyclic carbocyclic or heterocyclic radical having 5-13            ring members, or (ii), in the case where p is 0, a divalent            radical of formula -Q¹-X²— wherein X² is —O—, —S— or NR^(A)—            wherein R^(A) is hydrogen or optionally substituted C₁-C₃            alkyl, and Q¹ is an optionally substituted divalent mono- or            bicyclic carbocyclic or heterocyclic radical having 5-13            ring members,        -   Alk¹ and Alk² independently represent optionally substituted            divalent C₃-C₇ cycloalkyl radicals, or optionally            substituted straight or branched, C₁-C₆ alkylene, C₂-C₆            alkenylene, or C₂-C₆ alkynylene radicals which may            optionally contain or terminate in an ether (—O—), thioether            (—S—) or amino (—NR^(A)—) link wherein R^(A) is hydrogen or            optionally substituted C₁-C₃ alkyl; and    -   z is 0 or 1.

In another broad aspect the invention provides the use of a compound offormula (IA) or (IB) as defined above, or an N-oxide, salt, hydrate orsolvate thereof in the preparation of a composition for inhibiting theactivity of IKK, especially IKKβ, as well as diseases modulated by theNF-kB cascade.

The compounds with which the invention is concerned may be used for theinhibition of IKK, especially IKKβ, activity in vitro or in vivo.

Pharmaceutical compositions comprising a compound of the inventiontogether with one or more pharmaceutically acceptable carriers andexcipients, also form part of the invention.

In one aspect of the invention, the compounds of the invention may beused in the preparation of a composition for the treatment ofneoplastic/proliferative, autoimmune or inflammatory disease,particularly those mentioned above in which IKK, especially IKKβ,activity plays a role.

In another aspect, the invention provides a method for the treatment ofthe foregoing disease types, which comprises administering to a subjectsuffering such disease an effective amount of a compound of formula (IA)or (IB) as defined above.

Terminology

As used herein, the term “(C_(a)-C_(b))alkyl” wherein a and b areintegers refers to a straight or branched chain alkyl radical havingfrom a to b carbon atoms. Thus when a is 1 and b is 6, for example, theterm includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C_(a)-C_(b))alkylene radical” whereina and b are integers refers to a saturated hydrocarbon chain having froma to b carbon atoms and two unsatisfied valences.

As used herein the term “(C_(a)-C_(b))alkenyl” wherein a and b areintegers refers to a straight or branched chain alkenyl moiety havingfrom a to b carbon atoms having at least one double bond of either E orZ stereochemistry where applicable. The term includes, for example,vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

As used herein the term “divalent (C_(a)-C_(b))alkenylene radical” meansa hydrocarbon chain having from a to b carbon atoms, at least one doublebond, and two unsatisfied valences.

As used herein the term “(C_(a)-C_(b))alkynyl” wherein a and b areintegers refers to straight chain or branched chain hydrocarbon groupshaving from a to b carbon atoms and having in addition one triple bond.This term would include for example, ethynyl, 1-propynyl, 1- and2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

As used herein the term “divalent (C_(a)-C_(b))alkynylene radical”wherein a and b are integers refers to a divalent hydrocarbon chainhaving from a to b carbon atoms, and at least one triple bond.

As used herein the term “carbocyclic” refers to a mono-, bi- ortricyclic radical having up to 16 ring atoms, all of which are carbon,and includes aryl and cycloalkyl.

As used herein the term “cycloalkyl” refers to a monocyclic saturatedcarbocyclic radical having from 3-8 carbon atoms and includes, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyland cyclooctyl.

As used herein the unqualified term “aryl” refers to a mono-, bi- ortri-cyclic carbocyclic aromatic radical, and includes radicals havingtwo monocyclic carbocyclic aromatic rings which are directly linked by acovalent bond. Illustrative of such radicals are phenyl, biphenyl andnapthyl.

As used herein the unqualified term “heteroaryl” refers to a mono-, bi-or tri-cyclic aromatic radical containing one or more heteroatomsselected from S, N and O, and includes radicals having two suchmonocyclic rings, or one such monocyclic ring and one monocyclic arylring, which are directly linked by a covalent bond. Illustrative of suchradicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl,imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl,benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl,benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,indolyl and indazolyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in its non-aromatic meaningrelates to a mono-, bi- or tri-cyclic non-aromatic radical containingone or more heteroatoms selected from S, N and O, and to groupsconsisting of a monocyclic non-aromatic radical containing one or moresuch heteroatoms which is covalently linked to another such radical orto a monocyclic carbocyclic radical. Illustrative of such radicals arepyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl,benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl,ethylenedioxyphenyl, maleimido and succinimido groups.

A “divalent phenylene, pyridinylene, pyrimidinylene, or pyrazinyleneradical” is a benzene, pyridine, pyrimidine or pyrazine ring, with twounsatisfied valencies, and includes 1,3-phenylene, 1,4-phenylene, andthe following:

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four compatible substituents, each of which independently may be, forexample, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy, hydroxy(C₁-C₆)alkyl,mercapto, mercapto(C₁-C₆)alkyl, (C₁-C₆)alkylthio, phenyl, halo(including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy,nitro, nitrile (—CN), oxo, —COOH, —COOR^(A), —COR^(A), —SO₂R^(A),—CONH₂, —SO₂NH₂, —CONHR^(A), —SO₂NHR^(A), —CONR^(A)R^(B),—SO₂NR^(A)R^(B), —NH₂, —NHR^(A), —NR^(A)R^(B), —OCONH₂, —OCONHR^(A),—OCONR^(A)R^(B), —NHCOR^(A), —NHCOOR^(A), —NR^(B)COOR^(A), —NHSO₂OR^(A),—NR^(B)SO₂OH, —NR^(B)SO₂OR^(A),—NHCONH₂, —NR^(A)CONH₂, —NHCONHR^(B),—NR^(A)CONHR^(B), —NHCONR^(A)R^(B), or —NR^(A)CONR^(A)R^(B) whereinR^(A) and R^(B) are independently a (C₁-C₆)alkyl, (C₃-C₆) cycloalkyl ,phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or R^(A) andR^(B) when attached to the same nitrogen atom form a cyclic aminogroup(for example morpholino, piperidinyl, piperazinyl, ortetrahydropyrrolyl). An “optional substituent” may be one of theforegoing substituent groups.

As used herein the term “salt” includes base addition, acid addition andquaternary salts. Compounds of the invention which are acidic can formsalts, including pharmaceutically acceptable salts, with bases such asalkali metal hydroxides, e.g. sodium and potassium hydroxides; alkalineearth metal hydroxides e.g. calcium, barium and magnesium hydroxides;with organic bases e.g. N-methyl-D-glucamine, cholinetris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like. Those compounds (IA) and (IB)which are basic can form salts, including pharmaceutically acceptablesalts with inorganic acids, e.g. with hydrohalic acids such ashydrochloric or hydrobromic acids, sulphuric acid, nitric acid orphosphoric acid and the like, and with organic acids e.g. with acetic,tartaric, succinic, fumaric, maleic, malic, salicylic, citric,methanesulphonic, p-toluenesulphonic, benzoic, benzenesulfonic,glutamic, lactic, and mandelic acids and the like.

It is expected that compounds of the invention may be recovered inhydrate or solvate form. The term ‘solvate’ is used herein to describe amolecular complex comprising the compound of the invention and astoichiometric amount of one or more pharmaceutically acceptable solventmolecules, for example, ethanol. The term ‘hydrate’ is employed whensaid solvent is water.

Compounds of the invention which contain one or more actual or potentialchiral centres, because of the presence of asymmetric carbon atoms, canexist as a number of diastereoisomers with R or S stereochemistry ateach chiral centre. The invention includes all such diastereoisomers andmixtures thereof.

The term “ester” or “ester group” or “esterified carboxyl group” inconnection with substituent R₁ above means a group R_(x)O(C═O)— in whichR_(x) is the group characterising the ester, notionally derived from thealcohol R_(x)OH.

In the compounds of the invention, the variable substituents and groupswill now be discussed in more detail:

The Substituent R₇

R₇ is hydrogen or optionally substituted (C₁-C₆)alkyl, such as methyl,ethyl or n- or iso-propyl. Currently preferred is when R₇ is hydrogen.

The Ring A

Ring A is an optionally substituted divalent aryl or heteroaryl ring of5-13 atoms, such as a monocyclic 5- or 6-membered ring or a bicyclic5,6-, 6,6-, or 5,5-ring system. Examples include divalent phenylene,pyridinylene, pyrimidinylene, and pyrazinylene radicals. Currentlypreferred is 1,4-phenylene or 1,3-phenylene. Optional substituents inring A may be selected from, for example fluoro, chloro, methyl, andtrifluoromethyl.

The Group Z

The Group R₁ in Z

R₁ is a carboxylic acid group or an ester group which is hydrolysable byone or more intracellular carboxylesterase enzymes to a carboxylic acidgroup. Intracellular carboxylesterase enzymes capable of hydrolysing theester group of a compound of the invention to the corresponding acidinclude the three known human enzyme isotypes hCE-1, hCE-2 and hCE-3.Although these are considered to be the main enzymes, other enzymes suchas biphenylhydrolase (BPH) may also have a role in hydrolysing theester. In general, if the carboxylesterase hydrolyses the free aminoacid ester to the parent acid it will also hydrolyse the ester motifwhen covalently conjugated to the IKK inhibitor. Hence, the broken cellassay described herein provides a straightforward, quick and simplefirst screen for esters which have the required hydrolysis profile.Ester motifs selected in that way may then be re-assayed in the samecarboxylesterase assay when conjugated to the modulator via the chosenconjugation chemistry, to confirm that it is still a carboxylesterasesubstrate in that background.

Subject to the requirement that they be hydrolysable by intracellularcarboxylesterase enzymes, examples of particular ester groups R₁ includethose of formula —(C═O)OR₁₄ wherein R₁₄ is R₈R₉R₁₀C— wherein

-   -   (i) R₈ is hydrogen or optionally substituted        (C₁-C₃)alkyl-(Z¹)_(a)—[(C₁-C₃)alkyl]_(b)- or        (C₂-C₃)alkenyl-(Z¹)_(a)—[(C₁-C₃)alkyl]_(b)- wherein a and b are        independently 0 or 1 and Z¹ is —O—, —S—, or —NR₁₁— wherein R₁₁        is hydrogen or (C₁-C₃)alkyl; and R₉ and R₁₀ are independently        hydrogen or (C₁-C₃)alkyl-;    -   (ii) R₈ is hydrogen or optionally substituted        R₁₂R₁₃N—(C₁-C₃)alkyl- wherein R₁₂ is hydrogen or (C₁-C₃)alkyl        and R₁₃ is hydrogen or (C₁-C₃)alkyl; or R₁₂ and R₁₃ together        with the nitrogen to which they are attached form an optionally        substituted monocyclic heterocyclic ring of 5- or 6-ring atoms        or bicyclic heterocyclic ring system of 8 to 10 ring atoms, and        R₉ and R₁₀ are independently hydrogen or (C₁-C₃)alkyl-; or    -   (iii) R₈ and R₉ taken together with the carbon to which they are        attached form an optionally substituted monocyclic carbocyclic        ring of from 3 to 7 ring atoms or bicyclic carbocyclic ring        system of 8 to 10 ring atoms, and R₁₀ is hydrogen.

R₁ may be, for example, a methyl, ethyl, n- or iso-propyl, n-, sec- ortert-butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl,methoxyethyl, indanyl, norbornyl, dimethylaminoethyl, or morpholinoethylester group. Cyclopentyl or tert-butyl esters are currently preferred.

The Ring D in Z

When R is a group of formula (Y), examples of R include:

wherein R₁ is as defined and discussed above.

The Group R₆ in Z

R₆ may be optionally substituted C₁-C₆ alkyl, C₃-C₇ cycloalkyl, aryl orheteroaryl, for example methyl, ethyl, n-or isopropyl, cyclopropyl,cyclopentyl, cyclohexyl, phenyl, or pyridyl. In cases where macrophagespecificity is not required, R₆ may be hydrogen or —(C═O)R^(D), whereinR^(D) is optionally substituted (C₁-C₆)alkyl such as methyl, ethyl, n-orisopropyl, or n-, iso- or sec-butyl, (C₃-C₇)cycloalkyl such ascyclopropyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, thienyl,phenyl(C₁-C₆ alkyl)-, thienyl(C₁-C₆ alkyl)- or pyridyl(C₁-C₆ alkyl)-such as benzyl, 4-methoxyphenylmethylcarbonyl, thienylmethyl orpyridylmethyl.

R₆ may also be, for example —(C═O)OR^(D), or —(C═O)NHR^(D) wherein R^(D)is hydrogen or optionally substituted (C₁-C₆)alkyl such as methyl,ethyl, or n-or isopropyl.

For compounds of the invention which are to be administeredsystemically, esters with a slow rate of esterase cleavage arepreferred, since they are less susceptible to pre-systemic metabolism.Their ability to reach their target tissue intact is thereforeincreased, and the ester can be converted inside the cells of the targettissue into the acid product. However, for local administration, wherethe ester is either directly applied to the target tissue or directedthere by, for example, inhalation, it will often be desirable that theester has a rapid rate of esterase cleavage, to minimise systemicexposure and consequent unwanted side effects. If a carbon atom to whichthe group R is attached is unsubstituted, i.e. R is attached to amethylene (—CH₂)— radical, then the esters tend to be cleaved morerapidly than if that carbon is substituted, or is part of a ring systemsuch as a phenyl or cyclohexyl ring.

The Radical -L¹-Y¹—[CH₂]_(z)— in Z

This radical (or bond) arises from the particular chemistry strategychosen to link the amino acid ester motif R in substituent Z to the restof the molecule. Clearly the chemistry strategy for that coupling mayvary widely, and thus many combinations of the variables Y¹, L¹, and zare possible. However, when the inhibitor is bound to the enzyme at itsactive site, the amino acid ester motif generally extends in a directionaway from the enzyme, and thus minimises or avoids interference with thebinding mode of the inhibitor. Hence the precise combination of variablemaking up the linking chemistry between the amino acid ester motif andthe rest of the molecule will often be irrelevant to the primary bindingmode of the compound as a whole.

With the foregoing general observations in mind, taking the variablesmaking up the radical -L¹-Y¹—[CH₂]_(z)— in turn:

-   -   z may be 0 or 1, so that a methylene radical linked to the rest        of the molecule is optional;    -   Y¹ may be, for example, —NR₃—, —S—, —O—, —C(═O)NR₃—, —NR₃C(═O)—,        or —C(═O)O—, wherein R₃ is hydrogen or optionally substituted        C₁-C₆ alkyl such as —CH₂CH₂OH;    -   In the radical L¹, examples of Alk¹ and Alk² radicals, when        present, include —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,        —CH═CH—, —CH═CHCH₂—, —CH₂CH═CH—, CH₂CH═CHCH₂—, —C≡C—, —C≡CCH₂—,        CH₂C≡C—, and CH₂C≡CCH₂. Additional examples of Alk¹ and Alk²        include —CH₂W—, —CH₂CH₂W—, —CH₂CH₂WCH₂—, —CH₂CH₂WCH(CH₃)—,        —CH₂WCH₂CH₂—, —CH₂WCH₂CH₂WCH₂—, and —WCH₂CH₂— where W is —O—,        —S—, —NH—, —N(CH₃)—, or —CH₂CH₂N(CH₂CH₂OH)CH₂—. Further examples        of Alk¹ and Alk² include divalent cyclopropyl, cyclopentyl and        cyclohexyl radicals.    -   Alk¹ and Alk² when present may also be branched chain alkyl such        as —CH(CH₃)—, —C(CH₃)₂—, or in either orientation —CH₂CH(CH₃)—,        —CH₂C(CH₃)₂—.    -   In L¹, when n is 0, the radical is a hydrocarbon chain        (optionally substituted and perhaps having an ether, thioether        or amino linkage). Presently it is preferred that there be no        optional substituents in L¹. When both m and p are 0, L¹ is a        divalent mono- or bicyclic carbocyclic or heterocyclic radical        with 5-13 ring atoms (optionally substituted). When n is 1 and        at least one of m and p is 1, L¹ is a divalent radical including        a hydrocarbon chain or chains and a mono- or bicyclic        carbocyclic or heterocyclic radical with 5-13 ring atoms        (optionally substituted). When present, Q may be, for example, a        divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or        cyclohexyl radical, or a mono-, or bi-cyclic heterocyclic        radical having 5 to13 ring members, such as piperidinyl,        piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical, but        1,4-phenylene is presently preferred.    -   Specifically, in some embodiments of the invention, L¹, m and p        may be 0 with n being 1. In other embodiments, n and p may be 0        with m being 1. In further embodiments, m, n and p may be all 0.        In still further embodiments m may be 0, n may be 1 with Q being        a monocyclic heterocyclic radical, and p may be 0 or 1. Alk¹ and        Alk², when present, may be selected from —CH₂—, —CH₂CH₂—, and        —CH₂CH₂CH₂— and Q may be 1,4-phenylene.

Specific examples of the radical -L¹-Y¹—[CH₂]_(z)— include-L₁-Y¹—(CH₂)_(z)— in Z is —(CH₂)_(a)(O)_(d)(CH₂)_(a) wherein a is 1, 2or 3, b is 0, 1 or 2, and d is 0 or 1, —CH═CH—, or —CH₂CH═CH—.—CH═CHCH₂—, —C≡C—, —CH₂C≡C—, —C≡CCH₂—, —(CH₂)₃NH—, —CH₂C(═O)NH—,—CH₂CH₂C(═O)NH—,—CH₂C(O)O—, —CH₂S—, —CH₂CH₂C(O)O—, —(CH₂)₄NH—,—CH₂CH₂S—,

Specific compounds of the invention include those of the examplesherein, their salts, N-oxides, hydrates and solvates.

As mentioned above, the compounds with which the invention is concernedare inhibitors of IKK, especially IKKβ kinase activity, and aretherefore of use in the treatment of diseases modulated by IKK activityand the NF-kB cascade. Such diseases include neoplastic/proliferative,immune and inflammatory disease. In particular, uses of the compoundsinclude treatment of cancers such as hepatocellular cancer or melanoma,but including bowel cancer, ovarian cancer, head and neck and cervicalsquamous cancers, gastric or lung cancers, anaplasticoligodendrogliomas, glioblastoma multiforme or medulloblastomas; andtreatment of rheumatoid arthritis, psoriasis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, chronic obstructivepulmonary disease, asthma, multiple sclerosis, diabetes, atopicdermatitis, graft versus host disease, or systemic lupus erythematosus.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties. The orally administrable compositions may be in the form oftablets, capsules, powders, granules, lozenges, liquid or gelpreparations, such as oral, topical, or sterile parenteral solutions orsuspensions. Tablets and capsules for oral administration may be in unitdose presentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

The compounds of the invention may be administered in inhaled form.Aerosol generation can be carried out using, for example,pressure-driven jet atomizers or ultrasonic atomizers, preferably usingpropellant-driven metered aerosols or propellant-free administration ofmicronized active compounds from, for example, inhalation capsules orother “dry powder' delivery systems.

The active compounds may be dosed as described depending on the inhalersystem used. In addition to the active compounds, the administrationforms may additionally contain excipients, such as, for example,propellants (e.g. Frigen in the case of metered aerosols),surface-active substances, emulsifiers, stabilizers, preservatives,flavorings, fillers (e.g. lactose in the case of powder inhalers) or, ifappropriate, further active compounds.

For the purposes of inhalation, a large number of systems are availablewith which aerosols of optimum particle size can be generated andadministered, using an inhalation technique which is appropriate for thepatient. In addition to the use of adaptors (spacers, expanders) andpear-shaped containers (e.g. Nebulator®, Volumatic®), and automaticdevices emitting a puffer spray (Autohaler®), for metered aerosols, inparticular in the case of powder inhalers, a number of technicalsolutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or theinhalers for example as described EP-A-0505321).

For topical application to the eye, the drug may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentcan be dissolved in the vehicle.

The compounds of the invention may be used in conjunction with a numberof known pharmaceutically active substances. For example, the compoundsof the invention may be used with cytotoxics, HDAC inhibitors, kinaseinhibitors, aminopeptidase inhibitors, protease inhibitors, bcl-2antagonists, inhibitors of mTor and monoclonal antibodies (for examplethose directed at growth factor receptors). Preferred cytotoxicsinclude, for example, taxanes, platins, anti-metabolites such as5-fluoracil, topoisomerase inhibitors and the like. The medicaments ofthe invention comprising amino acid derivatives of formula (IA) or (IB),tautomers thereof or pharmaceutically acceptable salts, N-oxides,hydrates or solvates thereof therefore typically further comprise acytotoxic, an HDAC inhibitor, a kinase inhibitor, an aminopeptidaseinhibitor and/or a monoclonal antibody.

Further, the present invention provides a pharmaceutical compositioncomprising:

-   -   (a) an amino acid derivative of formula (IA) or (IB), or a        pharmaceutically acceptable salt, N-oxide, hydrate or solvate        thereof;    -   (b) a cytotoxic agent, an HDAC inhibitor, a kinase inhibitor, an        aminopeptidase inhibitor, a protease inhibitor, a bcl-2        antagonist, an inhibitor of mTor and/or a monoclonal antibody;        and    -   (c) a pharmaceutically acceptable carrier or diluent.

Also provided is a product comprising:

-   -   (a) an amino acid derivative of formula (IA) or (IB), or a        pharmaceutically acceptable salt, N-oxide, hydrate or solvate        thereof; and    -   (b) a cytotoxic agent, an HDAC inhibitor, a kinase inhibitor, an        aminopeptidase inhibitor, a protease inhibitor, a bcl-2        antagonist, an inhibitor of mTor and/or a monoclonal antibody,

for the separate, simultaneous or sequential use in the treatment of thehuman or animal body.

Synthesis

There are multiple synthetic strategies for the synthesis of thecompounds (I) with which the present invention is concerned, but allrely on known chemistry, known to the synthetic organic chemist. Thus,compounds according to formula (I) can be synthesised according toprocedures described in the standard literature and are well-known tothose skilled in the art. Typical literature sources are “Advancedorganic chemistry”, 4^(th) Edition (Wiley), J March, “ComprehensiveOrganic Transformation”, 2^(nd) Edition (Wiley), R. C. Larock, “Handbookof Heterocyclic Chemistry”, 2^(nd) Edition (Pergamon), A. R. Katritzky),review articles such as found in “Synthesis”, “Acc. Chem. Res.”, “Chem.Rev”, or primary literature sources identified by standard literaturesearches online or from secondary sources such as “Chemical Abstract” or“Beilstein”.

The compounds of the invention may be prepared by a number of processesgenerally described below and more specifically in the Exampleshereinafter. In the reactions described below, it may be necessary toprotect reactive functional groups, for example hydroxyl, amino andcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions [see for example Greene,T. W., “Protecting Groups in Organic Synthesis”, John Wiley and Sons,1999]. Conventional protecting groups may be used in conjunction withstandard practice. In some instances deprotection may be the final stepin the synthesis of a compound of general formula (IA) or (IB), and theprocesses according to the invention described herein after areunderstood to extend to such removal of protecting groups.

As mentioned above, the compounds with which the invention is concernedare inhibitors of the IkB family, namely IKK-α and IKK-β, and aretherefore of use in the treatment of cell proliferative disease, such ascancer, and in treatment of inflammation, in humans and other mammals.

Abbreviations

MeOH=methanol

EtOH=ethanol

IPA=isopropyl alcohol

EtOAc=ethyl acetate

DCM=dichloromethane

DMF=dimethylformamide

DME=dimethyl ether

DMSO=dimethyl sulfoxide

DMAP=dimethylamino pyridine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

FMOC=9-fluorenylmethoxycarbonyl

Na₂CO₃=sodium carbonate

HCl=hydrochloric acid

DIPEA=diisopropylethylamine

MP-CNBH₃=macroporous triethylammonium methylpolystyrene cyanoborohydride

BEMP=2-tbutylimino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine

NaH=sodium hydride

NaOH=sodium hydroxide

NaHCO₃=sodium hydrogen carbonate

HCl=hydrochloric acid

Pd/C=palladium on carbon

PdCl₂(dppf)=[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II).

EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

KOAc=potassium acetate

TBAI=tetrabutyl ammonium iodide

ml=millilitre(s)

g=gram(s)

mg=milligram(s)

mol=mole(s)

mmol=millimole(s)

Sat=saturated

LCMS=high performance liquid chromatography/mass spectrometry

NMR=nuclear magnetic resonance

Commercially available reagents and solvents (HPLC grade) were usedwithout further purification. Solvents were removed using a Buchi rotaryevaporator. Microwave irradiation was carried out using a CEM Discoverymodel set at 300 W. Purification of compounds by flash chromatographycolumn was performed using silica gel, particle size 40-63μ μm (230-400mesh) obtained from Fluorochem. Purification of compounds by preparativeHPLC was performed on a Agilent prep system using reverse phase Agilentprep-C18 columns (5 μm, 50×21.2 mm), gradient 0-100% B (A=water/0.1%ammonia or 0.1% formic acid and B=acetonitrile/0.1% ammonia or 0.1%formic acid) over 10 min, flow=28 ml/min, UV detection at 254 nm.

¹H NMR spectra were recorded on a Bruker 400 or 300 MHz AV spectrometerin deuterated solvents. Chemical shifts (δ) are in parts per million.Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60F₂₅₄ (Merck) plates and visualized using UV light.

Analytical HPLC/MS were obtained as follows: Agilent Prep-C18 Scalarcolumn, 5 μm (4.6×50 mm, flow rate 2.5 ml/min) eluting with a H₂O-MeCNgradient containing 0.1% v/v formic acid over 7 minutes with UVdetection at 254 nm. Gradient information: 0.0-0.5 min: 95% H₂O-5% MeCN;0.5-5.0 min; Ramp from 95% H₂O-5% MeCN to 5% H₂O-95% MeCN; 5.0-5.5 min:Hold at 5% H₂O-95% MeCN; 5.5-5.6 min: Hold at 5% H₂O-95% MeCN, flow rateincreased to 3.5 ml/min; 5.6-6.6 min: Hold at 5% H₂O-95% MeCN, flow rate3.5 ml/min; 6.6-6.75 min: Return to 95% H₂O-5% MeCN, flow rate 3.5ml/min; 6.75-6.9 min: Hold at 95% H₂O-5% MeCN, flow rate 3.5 ml/min;6.9-7.0 min: Hold at 95% H₂O-5% MeCN, flow rate reduced to 2.5 ml/min.Mass spectra were obtained using an Agilent multimode source in eitherthe positive (APCI+ESI⁺) or negative (APCI+ESI⁻) mode.

Examples of such methods that may be employed in the synthesis ofcompounds of general formula (IA) and (IB) are set out, but not limitedto the reactions shown in Schemes 1-10 below.

Scheme 1 illustrates the general synthetic route for the preparation ofthe examples described below, using traditional Suzuki chemistry tocouple the relevant boronate ester intermediates (2a-5c and 7a-12) withthe central thiophene core (Intermediates 1 and 13).

Scheme 2 illustrates the synthesis to Intermediate 2a.

Scheme 3 illustrates the synthesis to Intermediate 3a.

Scheme 4 illustrates the synthesis to Intermediate 5a.

Scheme 5 illustrates the synthesis to Intermediate 6a.

Scheme 6 illustrates the synthesis to Intermediates 8 and 11.

Scheme 7 illustrates the synthesis to Intermediate 9.

Scheme 8 illustrates the synthesis to Intermediate 10.

Scheme 9 illustrates the synthesis to Example 19.

Scheme 10 illustrates the synthesis to Intermediate 12.

Intermediates Intermediate 15-Bromo-2-(carbamoylamino)thiophene-3-carboxamide

The synthesis of Intermediate 1 highlighted by Stages 1-4 in Scheme 1 isdetailed within WO03104218.

Intermediate 2a CyclopentylN-(tert-butoxycarbonyl)-O-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-D-homoserinate

The synthesis of Intermediate 2a is detailed within Scheme 2 and fullexperimental details are shown below.

Stage 1—O[tent-Butyl(dimethyl)silyl]-D-homoserine

To a suspension of D-homoserine (1 g, 8.4 mmol) in acetonitrile (10 ml)at 0° C. was added 1,8-diazabicyclo[5.4.0]undec-7-ene (1.32 ml, 8.8mmol, 1.05 eq). tert-Butyl-dimethylsilyl chloride (1.33 g, 8.8 mmol,1.05 eq) was then added portionwise over 5 minutes and the reactionmixture allowed to warm to room temperature and stirred for 16 hrs. Awhite precipitate had formed which was filtered off and washed withacetonitrile before drying under vacuum. The title compound was isolatedas a white solid (1.8 g, 92%).

¹H NMR (400 MHz, DMSO), δ: 7.5 (1H, br s), 3.7 (1H, m), 3.35 (4H, bm),1.95 (1H, m), 1.70 (1H, m), 0.9 (9H, s), 0.1 (6H, s).

Stage2—N-(tert-Butoxycarbonyl)-O-[tert-butyl(dimethyl)silyl]-D-homoserine

The suspension of O-[tent-Butyl(dimethyl)silyl]-D-homoserine (1.8 g, 7.7mmol) in DCM (100 ml) at 0° C. was treated with triethylamine (2.15 ml,15.4 mmol, 2 eq) and di-tert-butyl dicarbonate (1.77 g, 8.1 mmol, 1.05eq). The reaction mixture was stirred at room temperature for 16 hoursfor complete reaction. The DCM was removed under reduced pressure andthe mixture was treated with ethyl acetate/brine. The ethyl acetatelayer was dried over magnesium sulphate and evaporated under reducedpressure. The crude product was taken forward without furtherpurification (2.53 g, 99%).

¹H NMR (400 MHz, CDCl₃), δ: 7.5 (1H, br s), 5.85 (1H, d, J=6.5 Hz), 4.3(1H, m), 3.75 (2H, m), 1.95 (2H, m), 1.40 (9H, s), 0.85 (9H, s), 0.1(6H, s).

Stage 3—CyclopentylN-(tert-butoxycarbonyl)-O-[tert-butyl(dimethyl)silyl]-D-homoserinate

To a solution ofN-(tert-butoxycarbonyl)-O-[tert-butyl(dimethyl)silyl]-D-homoserine (2.53g, 7.6 mmol) in DCM (50 ml) at 0° C. was added cyclopentanol (1.39 ml,15.3 ml, 2 eq), EDC (1.61 g, 8.4 mmol, 1.1 eq) and DMAP (0.093 g, 0.76mmol, 0.1 eq). The reaction mixture was stirred for 16 hours at roomtemperature before evaporation under reduced pressure. The crude residuewas dissolved in ethyl acetate (100 ml) and washed with 1M HCl, 1MNa₂CO₃ and brine. The organic layer was then dried over magnesiumsulphate and evaporated under reduced pressure. The product was purifiedby column chromatography using ethyl acetate/heptane (1:4) to give 2.24g, 73% yield of title compound.

LCMS purity 100%, m/z 402.5 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃), δ: 5.2 (1H,d, J=6.3 Hz), 5.15 (1H, m), 4.2 (1H, m), 3.6 (2H, m), 2.0 (1H, m),1.95-1.55 (9H, bm), 1.4 (9H, s), 0.85 (9H, s), 0.1 (6H, s).

Stage4—Cyclopentyl(2R)-4-hydroxy-2-[(tert-butoxycarbonyl)amino]butanoate

CyclopentylN-(tert-butoxycarbonyl)-O-[tert-butyl(dimethyl)silyl]-D-homoserinate(1.57 g, 3.9 mmol) was dissolved in acetic acid:THF:water (3:1:1, 100ml). The reaction mixture was stirred at 30° C. for 16 hours forcomplete reaction. Ethyl acetate (200 ml) was added and washed with 1MNa₂CO₃, 1M HCl and brine. The ethyl acetate extracts were dried overmagnesium sulphate and evaporated under reduced pressure to give theproduct as a clear oil which crystallised on standing (1.0 g, 95%).

LCMS purity 100%, m/z 310.3 [M+Na]⁺, ¹H NMR (400 MHz, CDCl₃), δ: 5.4(1H, d, J=6.5 Hz), 5.2 (1H, m), 4.4 (1H, m), 3.65 (2H, m), 2.15 (1H, m),1.9-1.55 (9H, bm), 1.45 (9H, s).

Stage 5—Cyclopentyl(2R)-4-bromo-2-[(tert-butoxycarbonyl)amino]butanoate

To a slurry of N-bromo succinimide (1.86 g, 10.4 mmol) in DCM (16.2 ml)was added a solution of triphenyl phosphine (2.56 g, 9.74 mmol) in DCM(7.2 ml). The solution was stirred for a further 5 minutes afteraddition. Pyridine (338 μl, 4.18 mmol) was added, followed by a solutionof cyclopentyl(2R)-4-hydroxy-2-[(tert-butoxycarbonyl)amino]butanoate(1.0 g, 3.48 mmol) in DCM (8.8 ml). The solution was stirred for 18 hrs,concentrated in vacuo and the residual solvent azeotroped with toluene(3×16 ml). The residue was triturated with diethyl ether (10 ml) andethyl acetate:heptane (1:9, 2×10 ml). The combined ether and heptanesolutions was concentrated onto silica and purified by columnchromatography using ethyl acetate/heptane (1:9-2:8) to provide 1.02 g(84% yield) of title compound.

¹H NMR (400 MHz, CDCl₃), δ: 5.3-5.05(2 H, m), 4.45-4.3(1H, m), 3.45 (2H,t, J=7.3 Hz), 2.50-2.30 (1H, m), 2.25-2.10 (1H, m), 1.95-1.60 (8H, b m),1.47 (9H, s).

Stage 6—Cyclopentyl O-(4-bromophenyl)-D-homoserinate

A solution of 4-bromophenol (0.593 g, 3.43 mmol) in DMF (5 ml) wascooled to 0° C. with an ice bath and NaH (0.137 g, 3.43 mmol) was addedin a single portion. The reaction was allowed to warm to RT, sonicatedbriefly and cooled down again to 0° C. A solution ofcyclopentyl(2R)-4-bromo-2-[(tert-butoxycarbonyl)amino]butanoate (1.2 g,3.43 mmol) in THF (10 ml) was then added dropwise, and the reactionallowed to warm to room temperature. After 1 hr the reaction was heatedto 50° C. and monitored by TLC for complete reaction. After 4 hrs, thereaction appeared to be complete and was allowed to cool to roomtemperature and poured onto a mixture of EtOAc and saturated NaHCO₃. Theorganic layer was collected, washed with 3 portions of water, brine andthen dried (MgSO₄), filtered and evaporated in vacuo. The residue stillcontained small amounts of 4-bromophenol which was removed by scavengingwith MP-carbonate (2 g) in DCM (7 ml), and the filtrate was evaporatedto give the product as a white solid (1.2 g, 79%). m/z 443 [M+H]⁺.

Stage 7—CyclopentylN-(tert-butoxycarbonyl)-O-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-D-homoserinate

A mixture of cyclopentyl O-(4-bromophenyl)-D-homoserinate (1.2 g, 2.71mmol), KOAc (0.346 g, 3.53 mmol), Bis[pinacolato]diboron (1.378 g, 5.43mmol) and PdCl₂(dppf) (0.198 g, 0.271 mmol) in DMSO was heated under anitrogen atmosphere at 65° C. and monitored by LC-MS for the formationof the product. After 1 hr the reaction was complete, hence the reactionmixture was cooled to room temperature and poured onto a mixture ofEtOAc and 1M HCl. The layers were separated, the organic layer washedwith water and brine, dried over magnesium sulphate, filtered andevaporated under reduced pressure. The residue was subjected to columnchromatography eluting with 5% to 10% EtOAc in hexanes (0.65 g, 49%).m/z 490 [M+H]⁺.

Intermediate 2b CyclopentylN-(tert-butoxycarbonyl)-O-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl-L-homoserinate

Synthesised via analogous methods to Intermediate 2a, using L-Homoserineat Stage 1 of Scheme 2. LCMS: m/z 490 [M+H]⁺.

Intermediate 3a CyclopentylN-(tert-butoxycarbonyl)-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-L-norvalinate

The synthesis of Intermediate 3a is detailed within Scheme 3 and fullexperimental details are shown below.

Stage 1: (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 5-benzyl ester1-cyclopentyl ester

To a solution of Boc-L-Glu(OBzl)-OH (15 g, 44.5 mmol) in dichloromethane(220 ml) in an ice-bath, was added cyclopentanol (4.8 ml, 53.3 mmol, 1.2eq), EDC (9.4 g, 48.9 mmol, 1.1 eq) and DMAP (543 mg, 4.4 mmol, 0.1 eq).The reaction mixture was allowed to warm to room temperature and stirredfor 12 hours for complete reaction. The reaction mixture was dilutedwith DCM (200 ml) and washed with 1M HCl, 1M Na₂CO₃ and brine. Theorganic layer was then dried over magnesium sulphate and evaporatedunder reduced pressure. The product was purified by columnchromatography using ethyl acetate/heptane (1:4) to give 12.4 g, 69%yield of title compound as a white solid.

¹H NMR (300 MHz, CDCl₃), δ: 7.38 (5H, m), 5.70 (1H, m), 5.10 (2H, s),5.05 (1H, m), 4.25 (1H, m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H,bm), 1.47 (9H, s).

Stage 2: (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 1-cyclopentylester

(S)-2-tert-Butoxycarbonylamino-pentanedioic acid 5-benzyl ester1-cyclopentyl ester (12.4 g, 30.5 mmol) was dissolved in EtOAc (200 ml)and purged with nitrogen before addition of 20% Pd(OH)₂ on carboncatalyst (1.3 g). The reaction flask was then purged with hydrogen gasfor a period of 5 minutes before leaving under a balloon of hydrogen for5 hours for complete reaction. The catalyst was removed by filtration,washing with 50 ml EtOAc and the combined mother liquors were evaporatedunder reduced pressure. The title compound was isolated as a clear oil(7.73 g, 85%) and required no further purification.

¹H NMR (300 MHz, CDCl₃), δ: 10.0 (1H, br s), 5.70 (2H, m), 4.28 (1H, m),2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, bm), 1.47 (9H, s).

Stage 3: (S)-2-tert-Butoxycarbonylamino-5-hydroxy-pentanoic acidcyclopentyl ester

Ethyl chloroformate (2.45 ml, 25.6 mmol, 1.2 eq) was added at −20° C. toa stirred solution of (S)-2-tert-butoxycarbonylamino-pentanedioic acid1-cyclopentyl ester (6.73 g, 21.4 mmol) and N-methyl morpholine (3.05ml, 27.8 mmol, 1.3 eq) in THF (50 ml). The reaction mixture became verythick with precipitation of a white solid. The reaction was thereforediluted further with THF (100 ml) to aid mixing and left stirring at−20° C. for 2 hours. The precipitated mass was filtered off and thefiltrate was added over a period of 20 minutes to a solution of sodiumborohydride (2.43 g, 64.1 mmol, 3 eq) in THF (20 ml) and water (5 ml) at0° C. The reaction mixture was allowed to stir to room temperature andleft for 4 hours for complete reaction. The mixture was acidified to pH5 with 1M HCl and the THF removed under reduced pressure. The aqueoussolution was extracted with EtOAc (3×100 ml) and dried over magnesiumsulphate. The product was purified by column chromatography (DCM-5%MeOH/DCM) and isolated as a clear oil (5.0 g, 78%).

¹H NMR (300 MHz, CDCl₃), δ: 5.20 (2H, m), 4.25 (1H, m), 3.65 (2H, m),2.00-1.57 (12H, bm), 1.47 (9H, s).

Stage 4—(S)-5-Bromo-2-tert-butoxycarbonylamino-pentanoic acidcyclopentyl ester

To a slurry of N-bromo succinimide (3.54 g, 19.9 mmol, 3 eq) in DCM (30ml) was added a solution of triphenyl phosphine (4.87 g, 18.8 mmol, 2.8eq) in DCM (15 ml). The solution was stirred for a further 5 minutesbefore addition of pyridine (644 μl, 7.96 mmol, 1.2 eq) and a solutionof (S)-2-tert-butoxycarbonylamino-5-hydroxy-pentanoic acid cyclopentylester (2.0 g, 6.64 mmol) in DCM (20 ml). The solution was stirred for 18hrs, concentrated in vacuo and the residual solvent azeotroped withtoluene (3×30 ml). The residue was triturated with diethyl ether (30 ml)and ethyl acetate:heptane (1:9, 2×30 ml). The combined ether and ethylacetate/heptane solutions was concentrated onto silica and purified bycolumn chromatography using ethyl acetate/heptane (1:9-2:8) to provide1.34 g (55% yield) of title compound as a clear oil.

¹H NMR (300 MHz, CDCl₃), δ: 5.25 (1H, m), 5.05 (1H, bd), 3.45 (2H, m),2.00-1.55 (12H, bm), 1.45 (9H, s).

Stage 5: Cyclopentyl5-(4-bromophenoxy)-N-(tert-butoxycarbonyl)-L-norvalinate

To a solution of 4-bromophenol (261 mg, 1.510 mmol) in acetonitrile (2ml) under nitrogen, was added BEMP (397 μl, 1.373 mmol). The mixture wasstirred at 50° C. for 30 minutes before being cooled to room temperatureand (S)-5-bromo-2-tert-butoxycarbonylamino-pentanoic acid cyclopentylester (500 mg, 1.373 mmol) added as a solution in acetonitrile (2 ml).The reaction was heated at 50° C. for a further 1 hour and then themixture was analysed by LCMS. The reaction was poured onto 2M Na₂CO₃(100 ml) and extracted with EtOAc (2×100 ml). The combined organiclayers were washed with 2M Na₂CO₃ (3×100 ml) and brine (100 ml), driedover magnesium sulphate, filtered and concentrated in vacuo to afford acolourless oil. MP-carbonate (1.0 g) was added to a solution of thecrude product in dichloromethane (3.4 ml) and the solution was leftovernight at room temperature. LCMS indicated complete removal of4-bromophenol and so the reaction was filtered to remove theMP-carbonate and the resin was washed with DCM (2×50 ml). The filtratewas concentrated to afford the crude product which was purified bycolumn chromatography eluting with 5% EtOAc in iso-hexane. Yield oftitle compound=435 mg, 0.953 mmol, 69.4% yield. m/z 456 and 458 [M+H]⁺.

Stage 6: CyclopentylN-(tert-butoxycarbonyl)-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-L-norvalinate

DMSO (1.6 ml) was added to a vial containing cyclopentyl5-(4-bromophenoxy)-N-(tert-butoxycarbonyl)-L-norvalinate (430 mg, 0.942mmol), Bis[pinacolato]diboron (479 mg, 1.884 mmol), potassium acetate(120 mg, 1.225 mmol) and PdCl₂(dppf) (77 mg, 0.094 mmol) under nitrogen.Nitrogen was bubbled through the solution for approximately five minutesthen the reaction was heated to 65° C. overnight. LCMS indicatedcompletion of reaction and so the reaction was cooled to roomtemperature and partitioned between ether and water. The layers wereseparated and the organic layer was washed with water and brine, thendried over magnesium sulphate and concentrated in vacuo. Purificationwas achieved by column chromatography eluting with 10% EtOAc iniso-hexane. Yield of title compound=380 mg, 0.755 mmol, 80% yield. m/z504 [M+H]⁺.

Intermediate 3b CyclopentylN-(tert-butoxycarbonyl)-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-D-norvalinate

Synthesised via analogous methods to Intermediate 3a, usingBoc-D-Glu(OBzl)-OH at Stage 1 of Scheme 3. m/z 504 [M+H]⁺.

Intermediate 4a CyclopentylN-(tert-butoxycarbonyl)-O-[3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-L-homoserinate

Synthesised by analogous methods to Intermediate 2b, using4-bromo-3-methyl-phenol at Stage 6 of Scheme 2. m/z 504 [M+H]⁺.

Intermediate 4b CyclopentylN-(tert-butoxycarbonyl)-O-[2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-L-homoserinate

Synthesised by analogous methods to Intermediate 2b, using4-bromo-2-methyl-phenol at Stage 6 of Scheme 2. m/z 504 [M+H]⁺.

Intermediate 4c CyclopentylN-(tert-butoxycarbonyl)-O-[3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-L-homoserinate

Synthesised by analogous methods to Intermediate 2b, using4-bromo-3-chloro-phenol at Stage 6 of Scheme 2. m/z 524 [M+H]⁺.

Intermediate 4d CyclopentylN-(tert-butoxycarbonyl)-O-[2-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-L-homoserinate

Synthesised by analogous methods to Intermediate 2b, using4-bromo-2-chloro-phenol at Stage 6 of Scheme 2. m/z 524 [M+H]⁺.

Intermediate 5aCyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

The synthesis of Intermediate 5a is detailed within Scheme 4 and fullexperimental details are shown below.

Stage 1: Cyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate

To a stirred solution of DMAP (0.057 g, 0.465 mmol) in DCM (20 ml) wasadded EDC (0.980 g, 5.11 mmol). A solution of Boc-L-allylglycine (1.0 g,4.65 mmol) in DCM (5 ml) was added and the mixture stirred for 20minutes at RT. Cyclopentanol (0.506 ml, 5.58 mmol) was then added andthe mixture stirred overnight at RT for complete reaction. Afterconcentration of the reaction solvent in vacuo, the residue wassubjected to column chromatography eluting with 6% EtOAc in hexanes.Yield=0.907 g, 3.20 mmol, 68.9% yield. m/z 284 [M+H]⁺.

Stage 2:Cyclopentyl(2S,4E)-5-(4-bromophenyI)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate

A solution ofcyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate (0.88 g,3.11 mmol), 1-bromo-4-iodobenzene (0.966 g, 3.42 mmol), TBAI (1.262 g,3.42 mmol) and NaHCO₃ (0.783 g, 9.32 mmol) in acetonitrile (10 ml) waspurged with N₂ and palladium acetate (0.070 g, 0.311 mmol) was added.The reaction was then heated at 70° C. overnight. LCMS showed incompleteconversion and so the reaction was purged with nitrogen again and afurther 0.05 eq (35 mg) of Pd(OAc)₂, 0.25 eq (460 mg) of TBAI and 0.25eq of NaHCO₃ (65 mg) was added and stirred for another 24 hr at 70° C.After cooling to room temperature, the solvent was removed under reducedpressure. The crude residue was absorbed onto silica and purified bycolumn chromatography eluting with 10% EtOAc in iso-hexane. Yield=715mg, 1.631 mmol, 52.5% yield. LCMS purity>90%: m/z 438 and 440 [M+H]⁺.

Stage 3:Cyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

DMSO (4 ml) was added tocyclopentyl(2S,4E)-5-(4-bromophenyl)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate(700 mg, 1.597 mmol), Bis[pinacolato]diboron (811 mg, 3.19 mmol),potassium acetate (204 mg, 2.076 mmol) and PdCl₂(dppf) (130 mg, 0.160mmol) under a nitrogen atmosphere. Nitrogen was bubbled through forapproximately 5 minutes and then the reaction was heated at 65° C.overnight. LCMS indicated completion conversion and so the reaction wascooled to room temperature and partitioned between diethyl ether andwater. The layers were separated and the organic layer was washed withwater and brine, then dried over magnesium sulphate and concentrated invacuo. Purification was achieved by column chromatography eluting with6-10% EtOAc in iso-hexane. Yield=315 mg, 0.649 mmol, 40.6% yield. LCMSpurity>90%: m/z 486 [M+H]⁺.

Intermediate 5bCyclopentyl(2R,4E)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

Synthesised via analogous methods to Intermediate 5a, usingBoc-D-allylglycine at Stage 1 of Scheme 4. LCMS: m/z 486 [M+H]⁺.

Intermediate 5ctert-Butyl(2S,4E)-2-amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

The synthesis of Intermediate 5c is detailed below.

Stage 1: Fmoc-L-Allylglycine tert-butyl ester

To a solution of Fmoc-L-Allylglycine (4 g, 11.86 mmol) indichloromethane (120 ml) was added a solution oftert-butyl-2,2,2-trichloroacetimidate (4.24 ml, 23.71 mmol) incyclohexane (6 ml). Boron trifluoride etherate (1.502 ml, 11.86 mmol)was then added and the reaction mixture left to stir at RT overnight.LCMS indicated some starting material remained so a further 0.5 eq ofboron trifluoride etherate and 0.5 eq oftert-butyl-2,2,2-trichloroacetimidate were added to the reactionmixture. After stirring for 4 hours, reaction was complete. To thereaction mixture was added saturated NaHCO₃ solution (100 ml). Theorganic layer was separated and the cloudy aqueous layer was extractedwith a further two portions of dichloromethane (2×30 ml). Combinedorganics were washed sequentially with saturated NaHCO₃ solution andbrine before being dried (MgSO₄) and concentrated in vacuo to afford anopaque oil. The crude product was purified by column chromatography,eluting with 5% ethyl acetate in isohexane. Yield=3.4 g, 8.64 mmol, 58%.

Stages 2 and 3 follow similar experimental details to Stages 2 and 3 inthe synthesis of Intermediate 5a. The 9-fluorenylmethoxycarbonylprotecting group is conveniently removed using the reaction conditionsemployed in Stage 3. LCMS: m/z 374 [M+H]⁺.

Intermediate 6a CyclopentylN-(tert-butoxycarbonyl)-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-L-norvalinate

The synthesis of Intermediate 6a is detailed within Scheme 5 and fullexperimental details are shown below.

Stage 1:Cyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}pent-4-enoate

To a mixture ofcyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate(Intermediate 5a) (810 mg, 1.669 mmol), Intermediate 1 (401 mg, 1.517mmol) and tetrakis(triphenylphosphine) Pd catalyst (175 mg, 0.152 mmol)was added DME (8 ml) followed by a solution of sat. NaHCO₃ (3 ml). Themixture was placed in a preheated oil bath at 80° C. After 4 hrs thereaction was judged to be complete by LCMS. The mixture was cooled toRT, absorbed onto silica and subjected to column chromatography elutingwith 5% MeOH in DCM. Yield=0.5 g, 0.857 mmol, 56.5% yield. LCMS purity93%: m/z 543 [M+H]⁺, 541 [M−H]⁺.

Stage 2: CyclopentylN-(tert-butoxycarbonyl)-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-L-norvalinate

Hydrogen gas was bubbled through a suspension of Wilkinson's catalyst(767 mg, 0.829 mmol) in IPA (10 ml) and toluene (5 ml). After 5 minutes,a solution ofcyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}pent-4-enoate(300 mg, 0.553 mmol) in IPA (10 ml) was added. The mixture was keptunder an atmosphere of hydrogen and placed in a preheated oil bath at80° C. After 4 hrs the reaction was judged to be complete by LC-MS. Themixture was filtered whilst still hot and the filtrate evaporated invacuo. The residue was subjected to column chromatography eluting with4% MeOH in DCM. Yield=120 mg, 0.198 mmol, 36% yield. LCMS purity 90%:m/z 543 [M−H]⁺.

Intermediate 6b CyclopentylN-(tert-butoxycarbonyl)-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-norvalinate

Synthesised via analogous methods to Intermediate 6a, using Intermediate5b at Stage 1 of Scheme 5. m/z 545 [M+H]⁺.

Intermediate 7aCyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-[3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

Synthesised by analogous methods to Intermediate 5a, using4-bromo-3-chloro-iodobenzene at Stage 2 of Scheme 4. m/z 520 [M+H]⁺.

Intermediate 7bCyclopentyl(2S,4E)-2-[(tert-butoxycarbonyl)amino]-5-[2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

Synthesised by analogous methods to Intermediate 5a, using4-bromo-2-methyl-iodobenzene at Stage 2 of Scheme 4. m/z 500 [M+H]⁺.

Intermediate 8Cyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]bent-4-ynoate

The synthesis of Intermediate 8 is detailed within Scheme 6 and fullexperimental details are shown below.

Stage 1: Cyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoate

To a solution of Boc-L-propargyl glycine (5 g, 23.45 mmol) in DCM (100ml) was added cyclopentanol (3.03 g, 35.2 mmol), EDC (4.93 g, 25.8 mmol)and DMAP (0.286 g, 2.345 mmol). The mixture was stirred overnight at RT.The solvent was removed under reduced pressure and the residue wassubjected to column chromatography eluting with 7 to 12% EtOAc inhexanes. Yield=5.5 g, 19.55 mmol, 83% yield.

Stage 2:Cyclopentyl(2S)-5-(4-bromophenyl)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoate

To a mixture ofcyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoate (2.45 g,8.72 mmol) and 4-iodo-bromobenzene (2.96 g, 10.46 mmol) in diethyl ether(4 ml) was added copper iodide (0.166 g, 0.872 mmol) and PdCl₂(PPh₃)₂(0.306 g, 0.436 mmol). Diethylamine (5.43 ml, 52.3 mmol) was then addedand the mixture stirred at RT. After 2 hrs, LCMS showed completereaction. The mixture was poured onto water and extracted with diethylether. The combined organic layers were washed with water and brine,dried over magnesium sulphate, filtered and evaporated in vacuo. Thecrude residue was subjected to column chromatography eluting with 5 to10% EtOAc in hexanes. Yield=2.9 g, 6.51 mmol, 75% yield. LCMS purity98%: m/z 437 [M+H]⁺.

Stage 3:Cyclopentyl(2S)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-ynoate

To a mixture ofcyclopentyl(2S)-5-(4-bromophenyI)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoate(2.9 g, 6.65 mmol), Bis[pinacolato]diboron (2.025 g, 7.98 mmol),PdCl₂(dppf) (0.246 g, 0.332 mmol) and potassium acetate (0.978 g, 9.97mmol) was added DMSO (10 ml). The mixture was purged with nitrogen andplaced in a preheated oil bath at 80° C. for 12 hrs. The mixture waspoured onto water and extracted with diethyl ether. The combined etherextracts were washed with water (×3) and brine (×2), dried overmagnesium sulphate, filtered and evaporated in vacuo. The residue wassubjected to column chromatography eluting with 5 to 10% EtOAc inhexanes. Yield=2.1 g, 3.69 mmol, 55.6% yield. LCMS purity 85%: m/z 484[M+H]⁺.

Intermediate 9 CyclopentylN-(tert-butoxycarbonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-L-phenylalaninate

The synthesis of Intermediate 9 is detailed within Scheme 7 and followsanalogous experimental details already described above. m/z 460 [M+H]⁺.

Intermediate 10 CyclopentylN-(tert-butoxycarbonyl)-O-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]-L-homoserinate

The synthesis of Intermediate 10 is detailed within Scheme 8 and followsexperimental details shown below.

Stage 1: O-(4-bromobenzyl)-N-(tert-butoxycarbonyl)-L-homoserine

To a flask containing Boc-L-homoserine (5.181 g, 23.63 mmol)) in THF (50ml) was added sodium hydride (2.93 g, 73.3 mmol) and left stirring at 0°C. for 30 minutes. The reaction mixture was warmed to RT and left for 2hrs. 4-bromo-benzylbromide (11.81 g, 47.3 mmol) was then added as a THFsolution (15 ml) dropwise. The reaction was left at RT for 20 hrs. Thereaction was quenched with MeOH and the solvent removed in vacuo. Theresidue was dissolved in water and washed with Et₂O. The aqueous layerwas acidified with 2M HCl and exhaustively extracted with EtOAc. Theorganic layer was washed with water and brine, dried over magnesiumsulphate and concentrated in vacuo. The oily residue (11 g) was purifiedby column chromatography eluting with 50-100% EtOAc in hexanes andflushed with 10-50% MeOH in EtOAc. Yield=927 mg, 2.388 mmol, 10% yieldas a viscous oil. m/z 386/388 [M−H]⁺.

Stages 2 and 3 are described in analogous experimental details shownabove. m/z 504 [M+H]⁺.

Intermediate 11Cyclopentyl(2S,4Z)-2-[(tert-butoxycarbonyl)amino]-5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]pent-4-enoate

The synthesis of Intermediate 11 is detailed within Scheme 6 and followsexperimental details shown below.

Stage 4:Cyclopentyl(2S,4Z)-5-(4-bromophenyl)-2-[(tert-butoxycarbonyl)amino]pent-4-enoate

A mixture ofcyclopentyl(2S)-5-(4-bromophenyl)-2-[(tert-butoxycarbonyl)amino]pent-4-ynoate(750 mg, 1.719 mmol), quinoline (41 μl, 3.44 mmol), 5% Pd on CaCO₃ (500mg) was purged with nitrogen. EtOH (60 ml) was added and the mixture wasstirred under an atmosphere of H₂ at room temperature for 21 hrs.Because of incomplete reaction, the mixture was filtered through Celite,treated with fresh catalyst (500 mg) and stirred under an atmosphere ofH₂ at room temp for a further 18 hrs. The mixture was filtered throughCelite and the filtrate evaporated in vacuo to afford the crude productas a colourless oil. Column purification using 5% EtOAc/isohexaneafforded the product (640 mg, 1.416 mmol, 82% yield) as a clearcolourless oil. LCMS purity 97%: m/z 439 [M+H]⁺. ¹H NMR showed 8 wt %trans-alkene.

Stage 5 is described in analogous experimental details shown above. m/z486 [M+H]⁺.

Intermediate 12Cyclopentyl(2S)-4-{benzyl[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]amino}-2-[(tert-butoxycarbonyl)amino]butanoate

The synthesis of Intermediate 12 is detailed within Scheme 10 and fullexperimental details are shown below.

Stage 1: N-(4-Bromophenyl)-2,2,2-trifluoroacetamide

To an ice cold solution of 4-bromoaniline (3 g, 17.44 mmol) in DCM (40ml) was added triethylamine (3.53 g, 34.9 mmol) followed bytrifluoroacetic anhydride (2.67 ml, 19.18 mmol) by dropwise addition.After stirring at this temperature for 30 minutes, the reaction wasallowed to warm to RT and stirred for a further 1 hr. The mixture wasthen poured onto sat. NaHCO₃, the layers separated and the aqueous layerextracted with DCM. The combined organics extracts were washed withwater and brine, then dried (MgSO₄), filtered and evaporated in vacuo togive the product as an off-white solid (4.6 g, 17 mmol, 98% yield). LCMSpurity 95%: m/z 269 [M+H]⁺.

Stage 2:Cyclopentyl(2S)-4-[(4-bromophenyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate

Acetonitrile (40 ml) was added to a mixture ofN-(4-bromophenyl)-2,2,2-trifluoroacetamide (1.3 g, 3.71 mmol),cyclopentyl(2S)-4-bromo-2-[(tert-butoxycarbonyl)amino]butanoate (0.904g, 3.37 mmol) and K₂CO₃ (0.933 g, 6.75 mmol). The resulting suspensionwas heated to 60° C., DMF added (2 ml) and monitored for the formationof the product. After 6 days, the reaction was not complete; however thereaction was worked up to isolate any product formed. The solvent wasevaporated, the residue dissolved in a mixture of water and EtOAc andthe layers separated. The aqueous was extracted with a further portionof EtOAc and the combined organics, dried (MgSO₄), filtered andevaporated in vacuo to give a yellow oil (˜1.8 g). This was subjected tocolumn chromatography eluting with 10 to 15% EtOAc in hexanes to givethe title compound as a colourless oil (660 mg, 1.5 mmol, 44% yield).LCMS purity 95%: m/z 442 [M+H]⁺.

Stage 3:Cyclopentyl(2S)-4-[benzyl(4-bromophenyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate

To a mixture ofcyclopentyl(2S)-4-[(4-bromophenyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate(0.66 g, 1.495 mmol), benzyl bromide (1.023 g, 5.98 mmol) and K₂CO₃(0.620 g, 4.49 mmol) was added MeCN (15 ml) and DMF (1 ml). Thesuspension was heated overnight at 60° C. The reaction was then pouredonto a mixture of water and EtOAc, the layers separated and the aqueouslayer extracted with EtOAc. The combined organics were washed with waterand brine, then dried (MgSO₄), filtered and evaporated in vacuo to givean oil (˜1.5 g) which was subjected to column chromatography elutingwith 5 to 10% EtOAc in hexanes to give the product (550 mg, 1.1 mmol,69% yield). LCMS purity 95%: m/z 532 [M+H]⁺.

Stage 4:Cyclopentyl(2S)-4-{benzyl[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]amino}-2-[(tert-butoxycarbonyl)amino]butanoate

To a mixture of cyclopentyl(2S)-4-[benzyl(4-bromophenyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoate(0.55 g, 1.035 mmol), KOAc (0.152 g, 1.552 mmol), Bis[pinacolato]diboron(1.051 g, 4.14 mmol) and PdCl₂(dppf) (0.076 g, 0.103 mmol) was addedDMSO. The suspension was purged with nitrogen and heated to 70° C. andmonitored by LCMS for the formation of the product. After heatingovernight, the reaction was poured on to a mixture of sat. NaHCO₃ andEtOAc, the layers separated and the aqueous layer extracted with EtOAc.The combined organic layers were washed with water (×2) and brine, thendried (MgSO₄), filtered and evaporated in vacuo. The dark residue wassubjected to column chromatography eluting with 5 to 10% EtOAc inhexanes to give the product as a colourless oil (420 mg, 0.72 mmol, 70%yield). LCMS purity 95%: m/z 579 [M+H]⁺.

Intermediate 13 5-bromo-3-(carbamoylamino)thiophene-2-carboxamide

The synthesis of Intermediate 13 is detailed within WO2004063186.

Examples

The following examples illustrate the preparation of the specificcompounds of the invention, and the IKK inhibitory properties thereof:

Example 1 CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate

LC/MS purity 94%, m/z 447 [M+H]⁺, ¹H NMR (400 MHz, DMSO-d₆), δ: 10.9(1H, s), 8.5 (3H, br s), 7.6 (1H, br s), 7.5 (1H, s), 7.4 (2H, d, J=8.8Hz), 7.2 (1H, br s), 6.9 (2H, d, J=8.8 Hz), 5.1 (1H, m), 4.1 (3H, m),2.2 (2H, m), 1.9-1.5 (8H, m).

The synthesis of Example 1 is shown in Scheme 1.

Stage 5—CyclopentylN-(tert-butoxycarbonyl)-O-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate

DME (6 ml) was added to a mixture of cyclopentylN-(tert-butoxycarbonyl)-O-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-D-homoserinate(Intermediate 2a) (0.50 g, 1.022 mmol), Intermediate 1 (0.297 g, 1.124mmol) and NaHCO₃ (0.681 ml, 2.043 mmol). The reaction was flushedthoroughly with nitrogen and tetrakis(triphenylphosphine) Pd catalyst(0.118 g, 0.102 mmol) added. The reaction was heated to 80° C.overnight. Reaction was cooled to room temperature and poured onto amixture of DCM and sat NaHCO₃. The layers were separated and the aqueouslayer extracted with 2×DCM (30 ml). The combined organic layers weredried over magnesium sulphate, filtered, absorbed onto silica andsubjected to column chromatography eluting with 2% MeOH to 4% MeOH inDCM (198 mg, 36%). LCMS: m/z 548 [M+H]⁺.

Stage 6—CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate

A solution of CyclopentylN-(tert-butoxycarbonyl)-O-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate(0.24 g, 0.439 mmol) in THF (2 ml) was cooled to 0° C. 4M HCl in dioxane(2.195 ml, 8.78 mmol) was added and the solution allowed to warm to roomtemperature with stirring. After 10 minutes, a further equivalent of HClin dioxane (2.195 ml, 8.78 mmol) was added. The reaction was completeafter 2 hours and the solvents were evaporated under reduced pressureand the residue triturated with THF (10 ml). The solid was collected,washed with a large volume of diethyl ether and dried under vacuumovernight (201 mg, 95%). LCMS: m/z 447 [M+H]⁺.

The following examples were prepared in a similar manner to Example 1,using Intermediate 1 and the appropriate boronic ester, following theroute shown in Scheme 1.

Example Intermediate LCMS Number used R R₁₅ Name purity  2  2b

H Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}-L- homoserinate  94% purity: m/z 447 [M + H]⁺  3  3a

H Cyclopentyl 5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenoxy}-L- norvalinate  92% purity: m/z 461 [M + H]⁺  4  3b

H Cyclopentyl 5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenoxy}-D- norvalinate  96% purity: m/z 461 [M + H]⁺  5  4a

3-methyl Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]-3- methylphenyl}-L- homoserinate 100% purity: m/z 461 [M + H]⁺ 6  4b

2-methyl Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]-2- methylphenyl}-L- homoserinate 100% purity: m/z 461 [M + H]⁺ 7  4c

3-chloro Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]-3- chlorophenyl}-L- homoserinate 100% purity: m/z 482 [M + H]⁺ 8  4d

2-chloro Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]-2- chlorolphenyl}-L- homoserinate  98% purity: m/z 482 [M + H]⁺ 9  5a

H Cyclopentyl (2S,4E)- 2-amino-5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoate  92% purity: m/z 443 [M + H]⁺ 10  5b

H Cyclopentyl (2R,4E)- 2-amino-5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoate  98% purity: m/z 443 [M + H]⁺ 11  6a

H Cyclopentyl 5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}-L- norvalinate  92% purity: m/z 445 [M + H]⁺ 12  6b

H Cyclopentyl 5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}-D- norvalinate  98% purity: m/z 445 [M + H]⁺ 13  7a

3-chloro Cyclopentyl (2S,4E)- 2-amino-5-{4-[4- carbamoyl-5-(carbamoylamino)-2- thienyl]-3- chlorophenyl}pent-4- enoate  99% purity:m/z 478 [M + H]⁺ 14  7b

2-methyl Cyclopentyl (2S,4E)- 2-amino-5-{4-[4- carbamoyl-5-(carbamoylamino)-2- thienyl]-2- methylphenyl}pent-4- enoate 100% purity:m/z 457 [M + H]⁺ 15  8

H Cyclopentyl (2S)-2- amino-5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- ynoate  94% purity: m/z 441 [M + H]⁺ 16  9

H Cyclopentyl 4-[4- carbamoyl-5- (carbamoylamino)-2- thienyl]-L-phenylalaninate  99% purity: m/z 417 [M + H]⁺ 17 10

H Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]benzyl}-L- homoserinate  98% purity: m/z 461 [M + H]⁺ 18 11

H Cyclopentyl (2S,4Z)-2- amino-5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoate 100% purity: m/z 443 [M + H]⁺ ~15% transisomer by ¹H NMR 19* Example 2

H Cyclopentyl O-{4-[4- carbamoyl-5- (carbamoylamino)thiophen-2-yl]phenyl}-N- cyclohexyl-L- homoserinate  95% purity: m/z 529[M + H]⁺ 20** Example 10

H Cyclopentyl (2R,4E)- 5-{4-[4-carbamoyl-5- (carbamoylamino)thiophen-2-yl]phenyl}-2- (cyclohexylamino) pent-4-enoate 100% purity:m/z 525 [M + H]⁺ 21 12

H Cyclopentyl (2S)-2- amino-4-(benzyl{4-[4- carbamoyl-5-(carbamoylamino) thiophen-2- yl]phenyl}amino) butanoate  91% purity: m/z536 [M + H]⁺ 22  5c

H tert-Butyl (2S,4E)-2- amino-5-{4-[4- carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoate  97% purity: m/z 431 [M + H]⁺ *Thesynthesis of Example 19 is shown in Scheme 9 and detailed experimentalis shown below.

Stage 1: CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)thiophen-2-yl]phenyl}-N-cyclohexyl-L-homoserinate

Cyclohexanone (50.4 mg, 514 μmol) was added to a solution of Example 2(153 mg, 343 μmol) in THF (5 ml) and stirred at RT for 30 minutes.Sodium triacetoxyborohydride (363 mg, 1713 μmol) was then added slowly.After stirring at RT for 1 hr, LCMS indicated good conversion to theproduct. The reaction mixture was poured onto 0.5M HCl (5 ml) and DCM(10 ml) and allowed to stir for 10 minutes. The mixture was then pouredon to a large volume of sat. NaHCO₃, the layers separated and theaqueous layer extracted with two further portions of DCM. The combinedorganics extracts were dried over MgSO₄, filtered and evaporated invacuo to give a brown oil (˜300 mg). The crude product was purified bycolumn chromatography eluting with 5% MeOH in DCM to give the product asa white solid (65 mg, 123 μmol, 36%).

** The synthesis of Example 20 follows analogous experimental details toExample 19.

The following examples were prepared in a similar manner to Example 1,using Intermediate 13.

Example Intermediate LCMS Number used R R₁₅ Name purity 23 13 and 2b

H Cyclopentyl O-{4- [5-carbamoyl-4- (carbamoylamino)-2-thienyl]phenyl}-L- homoserinate 95% purity: m/z 447 [M + H]⁺ 24 13 and9

H Cyclopentyl 4-[5- carbamoyl-4- (carbamoylamino)- 2-thienyl]-L-phenylalaninate 98% purity: m/z 417 [M + H]⁺

NMR Data

Example Number NMR assignment 1 ¹H NMR (400 MHz, DMSO-d₆), δ: 10.9 (1H,s), 8.5 (3H, br s), 7.6 (1H, br s), 7.5 (1H, s), 7.4 (2H, d, J = 8.8Hz), 7.2 (1H, br s), 6.9 (2H, d, J = 8.8 Hz), 5.1 (1H, m), 4.1 (3H, m),2.2 (2H, m), 1.9-1.5 (8H, m). 5 ¹H NMR (400 MHz, DMSO- d₆), δ: 10.94(1H, s), 7.63 (1H, br. s), 7.16-7.26 (2H, m), 6.81 (1H, br. s), 6.74(2H, d, J = 8.6 Hz), 5.05 (1H, t, J = 6.1 Hz), 4.04 (3H, m), 3.27 (3H,s), 1.78 (2H, m), 1.49-1.61 (8H, m). 7 ¹H NMR (400 MHz, DMSO- d₆) δ:10.97 (1H, s), 7.66 (1H, br s), 7.45 (1H, s), 7.41 (1H, d, J = 8.8 Hz),7.22 (1H, br s), 7.06 (1H, d, J = 2.4 Hz), 6.93 (H, dd, J = 8.8 Hz),6.90 (1H, br s), 5.05 (1H, m), 4.03-4.15 (3H, m), 1.91-2.03 (1H, m),1.78 (2H, m), 1.48-1.60 (5H, m). 9 ¹H NMR (400 MHz, DMSO- d₆), δ: 10.96(1H, s), 7.69 (1H, s), 7.67 (1H, br s), 7.38-7.49 (2H, d, J = 8.8 Hz),7.32-7.38 (2H, d, J = 8.8 Hz), 7.25 (1H, br s), 6.94 (1H, br. s),6.33-6.40 (2H, m), 5.03 (1H, t, J = 5.9 Hz), 3.38 (1H, t, J = 6.4 Hz),2.33-2.44 (2H, m), 1.85-1.65 (2H, m), 1.40-1.60 (6H, m). 10 ¹H NMR (400MHz, DMSO- d₆), δ: 11.0 (1H, s), 7.7 (1H, s), 7.6 (1H, br s), 7.4 (2H,d, J = 8.4 Hz), 7.3 (2H, d, J = 8.4 Hz), 7.3 (1H, br s), 6.9 (2H, br s),6.4 (1H, d, J = 15.4 Hz), 6.2 (1H, m), 5.0 (1H, m), 3.4 (1H, t, J = 6.2Hz), 2.4 (2H, m), 2.1 (2H, br s), 1.7 (2H, m), 1.6-1.4 (6H, m). 11 ¹HNMR (400 MHz, DMSO- d₆), δ: 10.94 (1H, s), 7.63 (2H, s), 7.39 (2H, d, J= 8.3 Hz), 7.25 (1H, br. s), 7.15 (2H, d, J = 8.3 Hz), 6.91 (1H, br. s),5.02 (1 H, br. s), 5.02 (1H, t, J = 5.9 Hz), 3.23 (3H, m), 2.48- 2.58(2H, m), 1.75 (2H, m), 1.50-1.65 (8H, m). 12 ¹H NMR (400 MHz, DMSO- d₆),δ: 10.9 (1H, s), 7.6 (2H, br s), 7.4 (2H, d, J = 8.0 Hz), 7.3 (1H, brs), 7.2 (2H, d, J = 8.0 Hz), 6.9 (2H, br s), 5.0 (1H, m), 3.2 (1H, m),2.5 (2H, m), 1.8 (5H, m), 1.7-1.6 (2H, m), 1.6-1.5 (8H, m). 13 ¹H NMR(400 MHz, DMSO- d₆), δ: 11.0 (1H, s), 7.8 (1H, br s), 7.7 (1H, s), 7.5(2H, m), 7.4 (1H, m), 7.3 (1H, br s), 7.0 (2H, br s), 6.4 (2H, m), 5.1(1H, m), 3.4 (1H, t, J = 6.4 Hz), 2.4 (2H, m), 1.9-1.7 (4H, m), 1.7-1.4(6H, m). 14 ¹H NMR (400 MHz, DMSO- d₆), δ: 11.0 (1H, s), 7.7 (1H, s),7.7 (1H, br s), 7.5 (1H, d, J = 8 Hz), 7.3 (3H, m), 6.9 (2H, br s), 6.6(1H, d, J = 16.0 Hz), 6.1 (1H, m), 5.1 (1H, m), 3.4 (1H, t, J = 6.2 Hz),2.5 (2H, m), 2.3 (3H, s), 1.9-1.7 (4H, m), 1.7-1.4 (6H, m). 16 ¹H NMR(400 MHz, DMSO- d₆), δ: 10.95 (1H, s), 7.64 (2H, s), 7.38 (2H, d, J =8.3 Hz), 7.25 (1H, br. s), 7.16 (2H, d, J = 8.3 Hz), 6.91 (1H, br. s),4.97 (1 H, br. s), 4.96 (1H, t, J = 6.1 Hz), 3.46-3.51 (1H, m), 2.75(2H, d, J = 6.8 Hz), 1.75 (2H, m), 1.50-1.65 (8H, m).

Example 25O-{4-[4-Carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserine

LC/MS purity 94%, m/z 379 [M+H]⁺, ¹H NMR (400 MHz, DMSO-d₆), δ: 10.9(1H, s), 8.5 (3H, br s), 7.6 (1H, br s), 7.5 (1H, s), 7.29 (2H, d, J=8.8Hz), 7.2 (1H, br s), 6.9 (2H, d, J=8.8 Hz), 4.1 (3H, m), 2.2 (2H, m).

The synthetic route to Example 25 is detailed in Scheme 1 using Example1.

Stage 7

To a solution of CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate(Example 1) (0.05 g, 0.104 mmol) in THF was added lithium hydroxide (25mg, 1.035 mmol) in 1 ml water. The solution was allowed to stir at roomtemperature and monitored by LCMS for the formation of the product.After 2 hrs the reaction was judged to be complete. The THF wasevaporated under vacuum and the aqueous diluted with further water (2ml). Acetic acid was added dropwise until the pH was acidic (˜10 drops),the precipitate formed was collected via filtration and washed withwater (3 ml), ethanol (5 ml) and diethyl ether (10 ml). The product wasisolated as an off-white solid (29 mg, 74%).

The following examples were prepared in a similar manner to Example 25.

Example Example LCMS Number used R R₁₅ Name purity 26 2

H O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]phenyl}-L- homoserine 94% purity: m/z 447 [M + H]⁺ 27 3

H 5-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]phenoxy}-L- norvaline 96% purity: m/z 393 [M + H]⁺ 28 4

H 5-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]phenoxy}-D- norvaline 93% purity: m/z 393 [M + H]⁺ 29 5

3-methyl O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]-3-methylphenyl}-L- homoserine 100% purity: m/z 393 [M + H]⁺ 30 6

2-methyl O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]-2-methylphenyl}-L- homoserine 100% purity: m/z 393 [M + H]⁺ 31 7

3-chloro O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]-3-chlorophenyl}-L- homoserine 100% purity: m/z 414 [M + H]⁺ 32 8

2-chloro O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]-2-chlorophenyl}-L- homoserine 100% purity: m/z 414 [M + H]⁺ 33 9

H (2S,4E)-2-amino-5- {4-[4-carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoic acid  98% purity: m/z 375 [M + H]⁺ 34 10

H (2R,4E)-2-amino-5- {4-[4-carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoic acid 100% purity: m/z 375 [M + H]⁺ 35 11

H 5-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]phenyl}-L- norvaline 98% purity: m/z 377 [M + H]⁺ 36 12

H 5-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]phenyl}-D- norvaline 99% purity: m/z 377 [M + H]⁺ 37 13

3-chloro (2S,4E)-2-amino-5- {4-[4-carbamoyl-5- (carbamoylamino)-2-thienyl]-3- chlorophenyl}pent-4- enoic acid  99% purity: m/z 410 [M +H]⁺ 38 14

2-methyl (2S,4E)-2-amino-5- {4-[4-carbamoyl-5- (carbamoylamino)-2-thienyl]-2- methylphenyl}pent-4- enoic acid 100% purity: m/z 389 [M +H]⁺ 39 15

H (2S)-2-amino-5-{4- [4-carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- ynoic acid  95% purity: m/z 373 [M + H]⁺ 40 16

H 4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]-L- phenylalanine  97%purity: m/z 349 [M + H]⁺ 41 17

H O-{4-[4-carbamoyl-5- (carbamoylamino)-2- thienyl]benzyl}-L- homoserine 97% purity: m/z 393 [M + H]⁺ 42 18

H (2S,4Z)-2-amino-5- {4-[4-carbamoyl-5- (carbamoylamino)-2-thienyl]phenyl}pent-4- enoic acid 100% purity: m/z 375 [M + H]⁺ ~25%trans isomer by ¹H NMR 43 19

H O-{4-[4-carbamoyl-5- (carbamoylamino) thiophen-2-yl]phenyl}-N-cyclohexyl-L- homoserine  95% purity: m/z 461 [M + H]⁺ 44 20

H (2R,4E)-5-{4-[4- carbamoyl-5- (carbamoylamino)thiophen-2-yl]phenyl}-2- (cyclohexylamino) pent-4-enoic acid 100%purity: m/z 457 [M + H]⁺ 45 21

H (2S)-2-amino-4- (benzyl{4-[4- carbamoyl-5- (carbamoylamino)thiophen-2- yl]phenyl}amino) butanoic acid  92% purity: m/z 468 [M + H]⁺

The following examples were prepared in a similar manner to Example 25.

Example Example LCMS Number used R R₁₅ Name purity 46 23

H O-{4-[5-carbamoyl- 4- (carbamoylamino)- 2-thienyl]phenyl}-L-homoserine 92% purity: m/z 379 [M + H]⁺ 47 24

H 4-[5-carbamoyl-4- (carbamoylamino) 2-thienyl]-L- phenylalanine 98%purity: m/z 349 [M + H]⁺

Measurement of Biological Activity

IKKβ Enzyme Assay

The ability of compounds to inhibit IKKβ kinase activity was measured inan assay performed by Invitrogen (Paisley, UK). The Z′-LYTE™ biochemicalassay employs a fluorescence-based, coupled-enzyme format and is basedon the differential sensitivity of phosphorylated and non-phosphorylatedpeptides to proteolytic cleavage. The peptide substrate is labelled withtwo fluorophores—one at each end—that make up a FRET pair. In theprimary reaction, the kinase transfers the gamma-phosphate of ATP to asingle serine or threonine residue in a synthetic FRET-peptide. In thesecondary reaction, a site-specific protease recognizes and cleavesnon-phosphorylated FRET-peptides. Phosphorylation of FRET-peptidessuppresses cleavage by the Development Reagent. Cleavage disrupts FRETbetween the donor (i.e., coumarin) and acceptor (i.e. fluorescein)fluorophores on the FRET-peptide, whereas uncleaved, phosphorylatedFRET-peptides maintain FRET. A radiometric method, which calculates theratio (the Emission Ratio) of donor emission to acceptor emission afterexcitation of the donor fluorophore at 400 nm, is used to quantitatereaction progress.

The final 10 μL Kinase Reaction consists of 0.9-8.0 ng IKBKB (IKKβ), 2μM Ser/Thr 05 Peptide and ATP in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10mM MgCl₂, 1mM EGTA. The assay is performed at an ATP concentration at,or close to the Km. After the 60 minute Kinase Reaction incubation atroom temperature, 5 μL of a 1:128 dilution of Development Reagent isadded. The assay plate is incubated for a further 60 minutes at roomtemperature and read on a fluorescence plate reader.

Duplicate data points are generated from a ⅓ log dilution series of astock solution of test compound in DMSO. Nine dilutions steps are madefrom a top concentration of 10 μM, and a ‘no compound’ blank isincluded. Data is collected and analysed using XLfit software from IDBS.The dose response curve is curve fitted to model number 205 (sigmoidaldose-response model). From the curve generated, the concentration giving50% inhibition is determined and reported.

LPS-Stimulation of THP-1 Cells

THP-1 cells were plated in 100 μl at a density of 4×10⁴ cells/well inV-bottomed 96 well tissue culture treated plates and incubated at 37° C.in 5% CO₂ for16 hrs. 2 hrs after the addition of the inhibitor in 100 μlof tissue culture media, the cells were stimulated with LPS (E colistrain 005:B5, Sigma) at a final concentration of 1 μg/ml and incubatedat 37° C. in 5% CO₂ for 6 hrs. TNF-α levels were measured from cell-freesupernatants by sandwich ELISA (R&D Systems #QTA00B)

LPS-Stimulation of Human Whole Blood

Whole blood was taken by venous puncture using heparinised vacutainers(Becton Dickinson) and diluted in an equal volume of RPMI1640 tissueculture media (Sigma). 100 μl was plated in V-bottomed 96 well tissueculture treated plates. 2 hrs after the addition of the inhibitor in 100μl of RPMI1640 media, the blood was stimulated with LPS (E coli strain005:B5, Sigma) at a final concentration of 100 ng/ml and incubated at37° C. in 5% CO₂ for 6 hrs. TNF-α levels were measured from cell-freesupernatants by sandwich ELISA (R&D Systems #QTA00B)

IC50 values were allocated to one of three ranges as follows:

Range A: IC50<1000 nM

Range B: 1000 nM<IC50<5000 nM

Range C: IC50>5000 nM

NT=not tested

Results Table Inhibitor activity Inhibitor activity Example Inhibitoractivity versus THP-1 TNFα versus human whole Number versus IKKβ releaseblood TNFα release 1 A A B 2 A B NT 3 A B C 4 A B NT 5 A A C 6 A A C 7 AA C 8 A B C 9 A A A 10 A A A 11 A A B 12 A A NT 13 A A B 14 A A A 15 A BC 16 A A B 17 A B NT 18 A B B 19 A C NT 20 A B NT 21 A B NT 22 NT NT NT23 A B NT 24 A C NT 25 A NT NT 26 A NT NT 27 A NT NT 28 A NT NT 29 A NTNT 30 A NT NT 31 A NT NT 32 A NT NT 33 A NT NT 34 A NT NT 35 A NT NT 36A NT NT 37 A NT NT 38 A NT NT 39 A NT NT 40 A NT NT 41 A NT NT 42 A NTNT 43 A NT NT 44 A NT NT 45 A NT NT 46 A NT NT 47 A NT NT

Broken Cell Carboxylesterase Assay

Any given compound of the present invention wherein R₁ is an estergroup, may be tested to determine whether it meets the requirement thatit be hydrolysed by intracellular esterases, by testing in the followingassay.

Preparation of Cell Extract

U937 or HCT 116 tumour cells (˜10⁹) were washed in 4 volumes ofDulbeccos PBS (˜1 litre) and pelleted at 525 g for 10 min at 4° C. Thiswas repeated twice and the final cell pellet was resuspended in 35 ml ofcold homogenising buffer (Trizma 10 mM, NaCl 130 mM, CaCl₂ 0.5 mM pH 7.0at 25° C.). Homogenates were prepared by nitrogen cavitation (700 psifor 50 min at 4° C.). The homogenate was kept on ice and supplementedwith a cocktail of inhibitors at final concentrations of:

-   -   Leupeptin 1 μM    -   Aprotinin 0.1 μM    -   E64 8 μM    -   Pepstatin 1.5 μM    -   Bestatin 162 μM    -   Chymostatin 33 μM

After clarification of the cell homogenate by centrifugation at 525 gfor 10 min, the resulting supernatant was used as a source of esteraseactivity and was stored at −80° C. until required.

Measurement of Ester Cleavage

Hydrolysis of esters to the corresponding carboxylic acids can bemeasured using the cell extract, prepared as above. To this effect cellextract (˜30 μg/total assay volume of 0.5 ml) was incubated at 37° C. ina Tris-HCl 25 mM, 125 mM NaCl buffer, pH 7.5 at 25° C. At zero time theester (substrate) was then added at a final concentration of 2.5 μM andthe samples were incubated at 37° C. for the appropriate time (usually 0or 80 min). Reactions were stopped by the addition of 3× volumes ofacetonitrile. For zero time samples the acetonitrile was added prior tothe ester compound. After centrifugation at 12000 g for 5 min, sampleswere analysed for the ester and its corresponding carboxylic acid atroom temperature by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL).Chromatography was based on an AcCN (75×2.1 mm) column and a mobilephase of 5-95% acetonitrile in water/0.1% formic acid.

Table 1 presents data showing that several amino acid ester motifs,conjugated to various intracellular enzyme inhibitors by severaldifferent linker chemistries are all hydrolysed by intracellularcarboxyesterases to the corresponding acid.

TABLE 1 Hydrolysis Rate Range Preparation U937Cells of amino Structureof amino acid (pg/ ester ester conjugate R Linker mL/min) conjugate

~CH2CH2O~  100-  1000 WO2006117552

1000- 50000 WO2006117548

>50000 WO2006117549

~CH2CH2O~ >50000 WO2006117567

~CH2CH2O~ 1000- 50000 WO2006117567

~CH2~ 1000- 50000 WO2006117567

~CO~ >50000 WO2006117567

>50000 WO2006117549

>50000 WO2006117549

1. A compound of formula (IA) or (IB), or a salt, N-oxide, hydrate orsolvate thereof:

wherein R₇ is hydrogen or optionally substituted (C₁-C₆)alkyl; ring A isan optionally substituted aryl or heteroaryl ring of 5-13 ring atoms; Zis a radical of formula R-L¹-Y¹—(CH₂)_(z)—, wherein: R is a radical offormula (X) or (Y)

R₁ is a carboxylic acid group (—COOH), or an ester group which ishydrolysable by one or more intracellular esterase enzymes to acarboxylic acid group; R₆ is hydrogen; or optionally substituted C₁-C₆alkyl, C₃-C₇ cycloalkyl, aryl or heteroaryl or —(C═O)R₃, —(C═O)OR₃, or—(C═O)NR₃ wherein R₃ is hydrogen or optionally substituted (C₁-C₆)alkyl.Y¹ is a bond, —(C═O)—, —S(O₂)—, —C(═O)O—, —OC(═O)—, —(C═O)NR₃—,—NR₃(C═O)—, —S(O₂)NR₃—, —NR₃S(O₂)—, or —NR₃(C═O)NR₄—, wherein R₃ and R₄are independently hydrogen or optionally substituted (C₁-C₆)alkyl, L¹ isa divalent radical of formula -(Alk¹)_(m)(Q)_(n)(Alk²)_(p)- wherein m, nand p are independently 0 or 1, Q is (i) an optionally substituteddivalent mono- or bicyclic carbocyclic or heterocyclic radical having5-13 ring members, or (ii), in the case where p is 0, a divalent radicalof formula -Q¹-X²— wherein X² is —O—, —S— or NR^(A)— wherein R^(A) ishydrogen or optionally substituted C₁-C₃ alkyl, and Q¹ is an optionallysubstituted divalent mono- or bicyclic carbocyclic or heterocyclicradical having 5-13 ring members, Alk¹ and Alk² independently representoptionally substituted divalent C₃-C₇cycloalkyl radicals, or optionallysubstituted straight or branched, C₁-C₆ alkylene, C₂-C₆ alkenylene, orC₂-C₆ alkynylene radicals which may optionally contain or terminate inan ether (—O—), thioether (—S—) or amino (—NR^(A)—) link wherein R^(A)is hydrogen or optionally substituted C₁-C₃ alkyl; and z is 0 or
 1. 2. Acompound as claimed in claim 1 wherein R₇ is hydrogen.
 3. A compound asclaimed in claim 1 wherein ring A is optionally substituted1,4-phenylene or 1,3-phenylene.
 4. A compound as claimed in claim 1wherein optional substituents in ring A are selected from, fluoro,chloro, methyl, and trifluoromethyl.
 5. A compound as claimed in claim 1wherein R₁ is an ester group of formula —(C═O)OR₁₄ wherein R₁₄ isR₈R₉R₁₀C— wherein (i) R₈ is hydrogen or optionally substituted(C₁-C₃)alkyl-(Z¹)_(a)—[(C₁-C₃)alkyl]_(b)- or(C₂-C₃)alkenyl-(Z¹)_(a)—[(C₁-C₃)alkyl]_(b)- wherein a and b areindependently 0 or 1 and Z¹ is —O—, —S—, or —NR₁₁— wherein R₁1 ishydrogen or (C₁-C₃)alkyl; and R₉ and R₁₀ are independently hydrogen or(C₁-C₃)alkyl-; (ii) R₈ is hydrogen or optionally substitutedR₁₂R₁₃N—(C₁-C₃)alkyl- wherein R₁₂ is hydrogen or (C₁-C₃)alkyl and R₁₃ ishydrogen or (C₁-C₃)alkyl; or R₁₂ and R₁₃ together with the nitrogen towhich they are attached form an optionally substituted monocyclicheterocyclic ring of 5- or 6-ring atoms or bicyclic heterocyclic ringsystem of 8 to 10 ring atoms, and R₉ and R₁₀ are independently hydrogenor (C₁-C₃)alkyl-; or (iii) R₈ and R₉ taken together with the carbon towhich they are attached form an optionally substituted monocycliccarbocyclic ring of from 3 to 7 ring atoms or bicyclic carbocyclic ringsystem of 8 to 10 ring atoms, and R₁₀ is hydrogen.
 6. A compound asclaimed in claim 1 wherein R₁ is a methyl, ethyl, n- or iso-propyl, n-,sec- or tert-butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl,methoxyethyl, indanyl, norbornyl, dimethylaminoethyl, or morpholinoethylester group.
 7. A compound as claimed in claim 1 wherein R₁ is acyclopentyl or tert-butyl ester.
 8. A compound as claimed in claim 1wherein R₆ is hydrogen.
 9. A compound as claimed in claim 1 wherein-L¹-Y¹—(CH₂)_(z)— in Z is —(CH₂)_(a)(O)_(d)(CH₂)_(a) wherein a is 1, 2or 3, b is 0, 1 or 2, and d is 0 or 1, —CH═CH—, —CH₂CH═CH—, —CH═CHCH₂—,—C≡C—, —CH₂C≡C—, or —C≡CCH₂—.
 10. A compound as claimed in claim 1selected from the group consisting of CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-homoserinate,CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]-3-methylphenyl}-L-homoserinate,CyclopentylO-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]-3-chlorophenyl}-L-homoserinateCyclopentyl(2S,4E)-2-amino-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}pent-4-enoate,Cyclopentyl(2R,4E)-2-amino-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}pent-4-enoate,Cyclopentyl5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-L-norvalinate,Cyclopentyl5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]phenyl}-D-norvalinate,Cyclopentyl(2S,4E)-2-amino-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]-3-chlorophenyl}pent-4-enoate,Cyclopentyl(2S,4E)-2-amino-5-{4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]-2-methylphenyl}pent-4-enoate,and Cyclopentyl4-[4-carbamoyl-5-(carbamoylamino)-2-thienyl]-L-phenylalaninate, andsalts, N-oxides, hydrates or solvates thereof.
 11. A pharmaceuticalcomposition comprising a compound as claimed in claim 1 together withone or more pharmaceutically acceptable carriers and/or excipients. 12.A composition for inhibiting the activity of an IKK enzyme comprising acompound as claimed in claim
 1. 13. The composition as claimed in claim12 for the inhibition of IKKβ activity, ex vivo or in vivo. 14.composition for treatment of neoplastic/proliferative, immune orinflammatory disease comprising a compound as claimed in claim
 1. 15. Amethod of inhibiting the activity of an IKK enzyme comprising contactingthe enzyme with an amount of a compound as claimed in claim 1 effectivefor such inhibition.
 16. A method as claimed in claim 15 for theinhibition of IKKβ activity, ex vivo or in vivo.
 17. A method for thetreatment of neoplastic/proliferative, immune or inflammatory disease,which comprises administering to a subject suffering such disease aneffective amount of a compound as claimed in claim
 1. 18. The method asclaimed in claim 15 for the treatment of cancer cell proliferation. 19.The method as claimed in claim 15 for the treatment of hepatocellularcancer or melanoma.
 20. The method as claimed in claim 15 for thetreatment of bowel cancer, ovarian cancer, head and neck and cervicalsquamous cancers, gastric or lung cancers, anaplasticoligodendrogliomas, glioblastoma multiforme or medulloblastomas.
 21. Themethod as claimed in claim 15 for the treatment of rheumatoid arthritis,psoriasis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, chronic obstructive pulmonary disease, asthma, multiplesclerosis, diabetes, atopic dermatitis, graft versus host disease, orsystemic lupus erythematosus.